Elution electrophoresis



5 Sheets-Sheet 1 INVENTOR.

SAMUEL RAYMOND ATTORNEYS.

June 7, 1966 5, RAYMQND ELUTION ELECTROPHORESIS Filed Sept. 15, 1964 FIG. I

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June 7, 1966 s. RAYMOND 3,255,100

ELUTION ELECTROPHORESIS Filed Sept. 15, 1964 3 Sheets-Sheet 5 INVENTOR. SAMUEL RAYMOND IIl/IIIIIIIIIIIII FIG. 8

A TTORNEYS.

BY 4,4 5x444 M, 9 Owe/m4 United States Patent 3,255,100 ELUTION ELECTROPHORESIS Samuel Raymond, 4312 Osage Ave, Philadelphia 4, Pa. Filed Sept. 15, 1964, Ser. No. 396,664 7 Claims. (Cl. 204-180) This invention relates to improvements in the art of electrophoresis, and more particularly relates to a method and apparatus for elution convection electrophoresis.

Electrophoresis, generally, relates to the separation of a complex substance into its component fractions by procedures based upon the migration or mobility of electrically charged fractions in a direct current electricfield. With an electric field developed between .two spaced electrodes and a substance placed therein, the variously charged component fractions or constituents of the substance move or migrate toward the respective electrodes of opposite charge and the respective components will move with different mobilities, i.e., at different rates. Thus, after a suitable time separation of the various component fractions is accomplished. This migration and separation may take place on suitable support mediums such as a sheet of absorbent material-like filter paper or on gel such as acrylamide saturated with a buffer solution. Each component separated by the electrophoresis procedure is subject to a qualitative and quantitative analysis while on its support medium and has, therefore, provided a useful tool in laboratory analysis of various substances such as albumin, enzymes, hemoglobin, carbohydrates, blood serum, etc. as well as various inorganic ions.

Electrophoresis generally may be practiced by the apparatus shown in my prior patent, 3,047,489, or the prior patent to myself and my brother, 3,129,158, or other types of apparatus known in the art.

In the prior known electrophoresis,,each component may be analyzed while on its support medium and this type of electrophoresis may be considered as analytical electrophoresis. However, many times it is desirable and necessary to collect the isolated component fraction in its pure state and without other substances such as its support medium and without change in the component. The isolated component could then be put to further use. It is to this type of electrophoresis that this invention is directed and to distinguish it from the prior known analytical electrophoresis it is termed elution convection electrophoresis.

After the component fractions of a complex substance are separated by analytical electrophoresis while migrating in the direction of current between two electrodes while on a support medium, it is many times desirable to isolate the separated components from the support for further use. It is the object of this invention to provide a method and apparatus for preparing isolated components by themselves and without change by an electrophoresis procedure.

Other objects of the invention will be pointed out in the following description and claims and illustrated in the accompanying drawings, which disclose, by way of example, the principle of the invention and the best mode which has been contemplated of applying that principle.

In the drawings:

FIG. 1 is a schematic illustration of the apparatus of this invention placed in a buffer solution-containing tank -which is shown in section.

FIG. 2 is a side elevation of a separation grid used in the apparatus of this invention for keeping the separated components isolated from one another.

FIG. 3 is a sectional view of the grid taken along line 3-3 of FIG. 2.

FIG. 4 is a side elevation showing the reverse side ice of the' grid from that shown in FIG. 2 with a portionof the grid shown in section.

FIG. 5 is a detail view of a portion of the grid and adjacent components shown as detail A in FIG. 1.

FIG. 6 is an illustration of the separated components of a complex substance after practicing analytical electrophoresis thereon and illustrates how the support medium is rotated prior to practicing preparative electrophoresis of this invention.

FIG. 7 is a perspective exploded view of a preferred embodiment of the apparatus of this invention.

FIG. 8 is a sectional view taken through the assembled apparatus shown in FIG. 7.

FIG. 9 is a detail view of a portion of the separatio gridillustrating another method of practicing the invention.

In general, this invention provides a method and means for isolating and collecting pure components from their supporting medium after they have been separated by analytical electrophoresis. This is accomplished by placing the supporting electrophoresis medium with the separate zones of previously separated components thereon in a further electrophoresis cell with the direction of current in such cell at an angle to the plane of the supporting medium to remove the separated components from their support. After the separated components have been removed from their support and are migrating under the influence of the electric field, they are kept separated by a suitable separating block, their path of movement is intercepted by a substantially vertical dialysis membrane, then they accumulate at this membrane without passing through it, then the accumulated separated components on the membrane fall gravitationally in a convection current into separate collection receptacles.

Referring to the drawings for a more particular description of the invention, in FIG. 6 there is shown an electrophoresis support medium 10 which may be a slab of acrylamide gel or the like having thereon three zones of separated component fractions a, b, and c of a complex substance. These fractions having been previously separated from a complex substance in an electrophoresis process by passing direct current between electrodes generally in the direction of the arrows 12 for a suitable period of time. Such may be accomplished by means known in the art such as shown in my prior mentioned patents. The different components of the initial complex substance migrate at different rates under the influence of electrophoresis and they may be separated on the gel support using the apparatus of my prior Patent 3,047,489 or the process and disclosed apparatus of my Patent 3,129,158.

After the components a, b, and c are separated by electrophoresis into separate zones, the suporting medium which may be a gel slab 10 is removed from the apparatus and turned at an angle to the direction of original migration. Therefore, it is turned to the position as shown in the lower half of FIG. 6.- At this position, the separated components a, b, and c extend generally vertically. With the gel slab 10 in the position shown in the lower half of FIG. 6 it is placed in another electrophoresis cell in abuffer solution and an electric direct current field is applied in a direction generally normal or perpendicular to the plane of the slab 10.

As schematically illustrated in FIG. 1, the apparatus includes a tank 14 containing buffer solution 16 to a suitable level therein. A pair of wire grid electrodes 18 and 20 are connected to opposite poles 19 and 21 of a source of DC. so that a DC. field is established therebetween. The electrophoresis support medium 10 with the separated components a, b, and 0 extending generally vertically is placed against a separating grid 22 with the components facing and abutting one side of the grid. The grid has a plurality of holes 24 arranged in slightly overlapping vertical columns as shown in FIGS. 3 and 4 and extending completely through the grid 22. The pattern of holes 24 also slightly overlaps horizontally providing an arrangement similar to a honeycomb. This arrangement of the holes 24 will not leave any blank spaces between adjacent fractions. On the side of the grid opposite that where the gel slab is placed there are a plurality of component draining vertical grooves 26 as shown for example in FIG. 3. Similar vertical grooves 27 -are provided on the face of the grid adjacent the gel slab 10, and these groves allow for natural convection circulation of the buffer solution. Alternatively, grooves 26 could be much deeper to provide the same function. As a further alternative external curents of buffer solution may be applied eliminating the need for grooves 27. At the bottom of each vertical drain groove 26 there is a collecting receptacle 28. These collecting receptacles 28 may be provided in a separate block 29 which is secured to the grid block 22 as shown in FIGS. 4 and 5. On the back or grooved surface of the grid 22 there is a dialysis membrane 30 which allows the electric field and buffer solution to pass therethrough but will not pass the separated components.

Suitable pressing means are provided adjacent the electrodes 18 and and in the FIG. 1 embodiment these pressing means may be sponge blocks 32 and 34. Sponge block 32 presses the slab 10 against one face of the grid while pressing block 34 presses the dialysis membrane against the other side of the grid.

As an additional precaution and for sealing purposes a sealing means may be provided at the bottom of the dialysis membrane and this sealing means includes a rubber rod 36 and a groove 38 in the receptacle block. The pressing of the sponge 34 assures a seal at the area of the groove 38 in rod 36 so that separated fractions will not leak out from inside the membrane 30.

The assembly of electrodes, sponges, membrane matrix and gel slab are held together by suitable mechanical means not completely shown in the diagrammatical illustration of FIG. 1.

In operation, upon the application of a relative'low voltage D.C. curent to the electrodes, a field is established. and electrophoretically active material components a, b, and c (which are electronegative) migrate from the support 10 into the holes in the grid block 22 in a direction toward the positive electrode 20 with dialysis membrane 30 positioned in their path on the O1 posite face of the grid 22. The voltage between the electrodes is preferably less than 5 volts per cm. of electrode spacing to prevent the migrating components from sticking to the blocking membrane 30. The still separated pure components with the buffer solution flow by gravity downward in the vertical channels or grooves 26 until they are stopped by the seal at the bottom of the grooves and then they flow into the separate receptacles 28. Grooves 27 allow for recirculation of the buffer without the separated components. Thus, the sample receptacles 28 will contain the various separated components. To remove the components from the receptacles 28, the grid 22 is separated from the'receptacle block 29 or a pipette or the like may be inserted into the receptacle to remove the pure components.

FIG. 7 shows a preferred form of the apparatus in an exploded perspective view. In this form there is a suitable support 40 for a liquid holding tank 42. The tank has hollow side walls 44 and 46 having therein coolant passage 48 for the passage of cool-ant through tubes 50 and 52 for the purpose of temperature control of buffer solution in the tank. Sitting inside the tank is a generally vertical support box 54 having relieved portions 56 and 58 in the bottom of its walls to allow circulation of buffer solution (by external pump 59) and including a center slot or groove 69 in .both edges to support the hold the assembly and other suitable arrangements could' be used. Similarly, instead of the coolant passages 48 suitable external cooling means could be utilized. The separating grid block 62 may be the same or substantially the same as the block 22 in the FIG. 1 embodiment.

In the FIG. 7 embodiment, the pressing means for pressing the electrophoresis support medium against the matrix and for pressing the membrane against the matrix takes a different form, namely they are bags of dialysis membrane material such as a cellulose acetate film. More particularly, bags 64 and 66, shown partly cut away in FIG. 7 for the sake of clarity, completely enclose electrode assemblies 68 and 70. These bags are filled with buffer solution causing each bag to press against the matrix or separating grid 62 from both side faces thereof. However, prior to filling the bags the electrophoresis matrix or support medium 10 is placed against the face of the grid and is supported on support step 63 of the grid block 62.

The electrode assemblies 68 and 70 are substantially identical so only one will be described. Each electrode includes a frame 72 of Plexiglas or the like in which is embedded a grid type electrode such as a perforated stainless steel sheet 74, having suitable connecting terminals.

Circulating pump 59 circulates buffer solution to the inside of bags 64 and 66 through-suitable tubes 71 and 73. The solution flows into both bags simultaneously in parallel streams directed to the bottom of the bags. Then the solution flows upwardly and overflows the bags. The buffer solution which overflows from the bags into the tank 42 and is recirculated to the pump through passage 74 and this continuous recirculation of buflier solution is very beneficial as it accomplishes the-following:

(a) It keeps the bags filled to a height above the level of buffer in the tank 42. Without pumping, the liquid buffer would gradually dialyze out of the bags until the levels were equal inside and out.

(b) It remixes the anode and cathode buffer solution, thus neutralizing any pH or concentration changes which result from electrode reactions within the bags.

(0) It provides adequate heat transfer. Heat is generated by passage of the electric current through the electrodes, bags, gel slab, and separating grid. At normal operating currents and voltages, the heat is far too much to be removed by direct conduction; convection transfer of heat is impossible in the gel; the temperatures are not high enough for radiant heat transfer, and no evaporation can take place in the totally confined gel and matrix. Therefore, heat must be removed either by reducing the current and voltage (thereby prolonging the separating time unduly) or by pumping in cooled buffer and removing warmed buffer by overflow.

(d) Finally, continuous pumping allows us to use a construction in which there can be mass leaks from the bags 64, 66.. It is not easy to get leak-proof bags of dialysis membrane, and those presently available are not very large. But by using sheets of membrane held in place by gaskets, the apparatus can be designed for any size desired. Gaskets, however, entail leaks, and these, if not too great, can be overcome by sufficiently rapid pumping into the bags.

In operation of the FIG. 7 and 8 embodiment the electrophoretic support medium 10 with the zones of separated components, a, b, and 0 extending generally vertically thereon is placed against one side face of the grid block 62 as shown in FIGS. 7 and 8 and the grid is slid into groove 60 in the holding box 54 which in turn is placed in the tank 42. The two electrode assemblies are inserted together with their encircling bags 64 and 66 and the bags are filled to overflowing with buffer solution thereby covering the electrodes, the bags being made of dialysis membrane material to prevent the fractions from passing therethrough while allowing the buffer solution to pass. Then the entire tank 42 is filled with buffer solution to a level somewhat below the level of the buffer solution in the bag so that a hydrostatic head exists which acts to hold the face of one bag against the groove side of the matrix and also acts to press firmly but without excessive pressure on the gel slab on the other side of the grid. The pump 59 is started and the electrical connections are made to start the elution convection.

Another method of accomplishing preparative electrophoresis within the scope of this invention is illustrated in FIG. 9. In this method the electrophoresis gel slab with the initially separated components thereon is laid on a flat surface such as a table. The separating grid such as grid 22 or 62 is placed on top of the slab and aligned so that the vertical grooves 26 coincide with the zones or pattern of separated components on the gel slab 10. The grid 22 is then pressed into the gel slab 10. Because of the pliable nature of the gel, it is forced into the holes 24 of the grid. If the web of the grid between the holes is sufliciently narrow all of the gel can be squeezed into the hole thus subdividing the gel slab into a series of plugs filling or partially filling the holes 24 in the grid 22. This is accomplished by laying out the holes in a honeycomb pattern. Then, the grid carrying the plugs of gel is inserted into the vertical holder and a dialysis membrane is applied to the face of the separating matrix having the vertical grooves and is held thereagainst either by the flexible porous sponge 34 of FIG. 1 or by the bag 66 of FIG. 7 while the electric field is passed through the gel in a direction causing the components to migrate toward the dialysis membrane and the separated components then flow by gravity down the vertical grooves 26 into the collecting receptacles 28 as explained before.

While there have been shown and described and pointed out the fundamental novel features of the invention as applied to the preferred embodiment, it will be understood that various omissions and substitutions and changes in the form and details of the device illustrated and in its operation may be made by those skilled in the art without departing from the spirit of the invention. It is the intention therefore, to be limited only as indicated by the scope of the following claims.

I claim:

1. An apparatus for elution electrophoresis on components separated by analytical electrophoresis on a planar support medium, the apparatus comprising; a tank for containing a buffer solution, means for supporting the separated components on their planar support medium within the tank, a pair of generally planar electrodes sandwiching the faces of the support medium carrying the separated components, means for passing current from the electrodes to cause the components to migrate in buffer solution from their support medium towards one of the electrodes, mechanical means for fluid channelizing of separated components simultaneously, the mechanical means including a membrane permeable to an electric field but impermeable to the separated fractions and a dividing block, and means for simultaneously collecting the separated components.

2. A method of elution electrophoresis comprising;

(a) separating constituents of a composition by elec :trophoresis on a planar type support whereby different components migrate different distances on the support in a predetermined direction under the application of electric potential,

(b) removing the separated components from the planar support by subjecting them to an electric field in a buffer solution, the field directed generally at an angle to the plane of the support,

(c) keeping the removed components separate from each other, and

(d) collecting the separately removed individual components.

3. A method of elution-electrophoresis comprising separating components of a complex substance by electrophoresis on a planar type support whereby diflerent components of the substance migrate different distances to form a plurality of zones on the support under application of a DC. electric field, positioning the planar support with the separated components thereon so that the zones of the components extend generally vertically, removing the separated components from the planar support by subjecting them' to a DC. electric field while in a buffer solution with the field directed generally at an angle to the plane of the support, keeping the removed constituents separate from one another, and collecting the separate and now removed individual constituents.

4. A process for preparing pure concentrated component fractions from a complex substance of an electrophoretically active material, the process comprising; first, separating the fractions of the complex substance into generally parallel zones by electrophoresis in a planar electrophoretic medium, then electrophoretically eluting the separate fractions in the parallel zones while the fractions are transported in a buffer solution by an electric field passing through the planar .electrophoretic medium at an angle to the plane medium, keeping the eluted fractions in buffer solution separated from each other, and concentrating and collecting the eluted fractions in separate zones by transporting the eluted fractions by a separate current of buffer solution for each such fraction.

5. A process as defined in claim 4 further comprising interrupting electrophoretic migration of the eluted fractions during the elution step by a means permeable to the electric field and impermeable to the eluted fractions and external to the planar electrophoretic medium, and in which the concentrating and collecting of the eluted fractions is accomplished by buffer solution positively circulated flowing in a direction generally parallel to the separated zones.

6. A process as defined in claim 4 further comprising interrupting the electrophoretic migration ofthe eluted fractions during the elution step by a means permeable to the electric field and impermeable to the eluted fractions, and in which concentrating and collecting of the eluted fractions is accomplished by convection currents of buffer solution.

7. A process as defined in claim 6 in which the parallel zones are disposed in generally vertical position during the elution step,

OTHER REFERENCES Nature, vol. 202, No. 4936, pp. 1005-1006, June 1964.

References Cited by the Applicant UNITED STATES PATENTS 3,118,829 1/1964 Raymond. 3,129,158 4/1964 Raymond et al. 3,140,714 7/1964 Murphy et al.

OTHER REFERENCES Science, vol. 138, No. 3537, pp. 152-153, October 1962.

JOHN H. MACK, Primary Examiner.

E. ZAGARELLA, Assistant Examiner. 

4. A PROCESS FOR PREPARING PURE CONCENTRATED COMPOPHORETICALLY ACTIVE MATERIAL, THE PROCESS COMPRISING: FIRST, SEPARATING THE FRACTIONS OF THE CMPLEX SUBSTANCE INTO GENERALLY PARALLEL ZONES BY ELECTROPHORESIS IN A PLANAR ELECTROPHORETIC MEDIUM THEN ELECTROPHORETICALLY ELUTING THE SEPARATE FRACTIONS IN THE PARALLEL ZONES WHILE THE FRACTIONS ARE TRANSPORTED IN A BUFFER SOLUTION BY AN ELECTRIC FIELD PASSING THROUGH THE PLANAR ELECTROPHORETIC MEDIUM AT AN ANGLE TO THE PLANE MEDIUM, KEEPING THE ELUTED FRACTIONS IN BUFFER SOLUTION SEPARATED FROM EACH OTHER, AND CONCENTRATING AND COLLECTING THE ELUTED FRACTIONS IN SEPARATE ZONES BY TRANSPORTING THE ELUUTED FRACTIONS BY A SEPARATE CURRENT OF BUFFER SOLUTION FOR EACH SUCH FRACTION. 